Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis.

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.     

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.     

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.     

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.     

Interaction of glucosyltransferase from Streptococcus mutans with various glucans

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.     

Topological analysis of the membrane-bound glucosyltransferase, MdoH, required for osmoregulated periplasmic glucan synthesis in Escherichia coli.

The MdoH protein is essential for synthesis of the osmoregulated periplasmic glucans, known as membrane-derived oligosaccharides (MDOs), in Escherichia coli. Mutants lacking MdoH are deficient in a glucosyltransferase activity assayed in vitro. The MdoH protein is the product of the second gene of an operon, and it has been shown to span the cytoplasmic membrane. The MdoH protein comprises 847 amino acids and is poorly expressed as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have experimentally measured the topological organization of MdoH within the membrane by construction of fusions to beta-lactamase as a reporter. Analysis of 51 different MdoH-beta-lactamase fusions suggested that the MdoH protein crosses the cytoplasmic membrane eight times, with the N and C termini in the cytoplasm. Moreover, a 310-amino-acid domain is present in the cytoplasm between the second and third transmembrane segments. It was deduced from the measurement of the MDO biosynthetic activity of truncated or fused MdoH proteins that almost all the C-terminal residues are necessary for this activity. The model of the MdoH protein in the membrane suggests that this protein could be directly involved in the translocation of nascent polyglucose chains to the periplasmic space.     

Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharide

A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-specific polysaccharide biosynthesis. Correct stereochemistry of alpha- and beta-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.     

Chemoenzymatic synthesis of polyprenyl phosphates

Polyprenyl phosphates, including undecaprenyl phosphate and dolichyl phosphate, are essential intermediates in several important biochemical pathways including N-linked protein glycosylation in eukaryotes and prokaryotes and prokaryotic cell wall biosynthesis. Herein, we describe the evaluation of three potential undecaprenol kinases as agents for the chemoenzymatic synthesis of polyprenyl phosphates. Target enzymes were expressed in crude cell envelope fractions and quantified via the use of luminescent lanthanide-binding tags (LBTs). The Streptococcus mutans diacylglycerol kinase (DGK) was shown to be a very useful agent for polyprenol phosphorylation using ATP as the phosphoryl transfer agent. In addition, the S. mutans DGK can be coupled with two Campylobacter jejuni glycosyltransferases involved in N-linked glycosylation to efficiently biosynthesize the undecaprenyl pyrophosphate-linked disaccharide needed for studies of PglB, the C. jejuni oligosaccharyl transferase.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic β-(1,2)-Glucan Biosynthesis

The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.     

Synthesis of 2-deoxy-2-fluoro analogs of polyprenyl beta-D-arabinofuranosyl phosphates

Described is the synthesis of polyprenyl 2-deoxy-2-fluoro-beta-D-arabinofuranosyl phosphate derivatives, including an analog of decaprenyl beta-D-arabinofuranosyl phosphate, the donor species used by the arabinosyltransferases involved in mycobacterial cell-wall biosynthesis. The targets were synthesized via a route involving the synthesis of a protected beta-D-arabinofuranosyl phosphate derivative, its coupling with a polyprenyl trichloroacetimidate, and then deprotection of the resulting product. The use of arabinofuranosyl phosphates with the monosaccharide hydroxyl groups protected as either silyl ethers or benzoate esters was explored. Although the coupling yields between the phosphate and polyprenyl trichloroacetimidates were comparable with either type of protecting group, access to the benzoyl-protected derivative was more efficient and therefore gave the products in higher overall yield.     

Proteins containing reductively aminated disaccharides: immunochemical characterization.

Various polyprenyl phosphates were prepared by chemical phosphorylation of native and partially hydrogenated polyprenols. They were tested as lipid acceptors of sugars from nucleoside diphosphate sugars using a microsomal preparation from rat liver and membrane preparations from B. stearothermophilus, S. typhimurium, and Sh. flexneri. With the microsomal glycosyl transferase system, a demand for saturation of the α-isoprene residue of polyprenyl phosphate was observed; the chain length and cis/trans configuration of polyprenyl radical were less important. With bacterial glycosyl transferases, a demand for the unsaturated α-isoprene residue was observed. In B. stearothermophilus, the rate of synthesis of polyprenyl monophosphate glucose did not depend on the chain length of fully unsaturated polyprenyl phosphate. In S. typhimurium, C₅₅-polyprenyl phosphate was the most effective precursor of polyprenyl diphosphate galactose.     

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.     

Membrane properties of branched polyprenyl phosphates, postulated as primitive membrane constituents

We have postulated earlier that the highly branched isoprenoid alkanes, which are distributed widely in many sediments, may have been derived from the corresponding branched polyprenyl phosphates, potentially present in biomembranes in primitive organisms. These polyprenyl-branched polyprenyl phosphates might be derived by a simple alkylation from non-substituted polyprenyl phosphates, which we postulate to be the precursors of all membrane terpenoids. We have now synthesized a series of 6-(poly)prenyl-substituted polyprenyl phosphates and studied the formation of vesicles from these phosphates, as a function of the substituted-chain length, the position of the double bond, and pH. Nine of the branched polyprenyl phosphates containing 20-30 C-atoms do form vesicles at a 'physiological' pH; the lipophilicity/hydrophilicity ratio is as expected an important factor. We have also studied the water permeability through membranes of these branched polyprenyl phosphate vesicles by our stopped-flow/light-scattering method. These highly branched polyprenyl phosphates can more effectively reduce the water permeability than non-substituted polyprenyl phosphates: the vesicles formed by the former are more stable against mechanical stress. This reinforces our hypothesis about the origin of the sedimentary polyprenyl-substituted polyprene hydrocarbons.     

Biosynthesis of carbohydrate-containing polymers in plants. I. Products formed with enzyme preparation from clover seedlings. Characterization of polyprenylphosphosugars

Membrane preparations from clover seedlings catalyzed the incorporation of monosaccharide residues from GDPMan, UDPGlc and UDPGal into glycolipids, lipid-oligosaccharides and polymers. The lipid-oligosaccharides were shown to be alpha-dihydropolyprenyl pyrophosphate derivatives. Incorporation of mannose residues into the lipid-oligosaccharides and the polymers was significantly stimulated by addition of UDPGlc, GDPGlc, UDPGal but not by UDPGlcNAc. Exogenic polyprenyl phosphates also stimulated the process; the formation of moraprenyl pyrophosphate oligosaccharides was demonstrated after addition of moraprenyl phosphate. The lipid-oligosaccharides were precursors of the polymers which were shown to be mainly glycoproteins. A solubilized preparation of mannosyl transferase from clover membranes was obtained and some properties of the enzyme were studied.     

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.     

Novel fluorescent analogues for transmembrane movement study of polyprenyl phosphates

In order to investigate the transmembrane movement of polyprenyl phosphate across biological membranes, NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled polyprenyl phosphate analogues were prepared. These analogues proved to be possible tools for a direct observation of the transmembrane flip-flop movement of polyprenyl phosphates by use of a sodium dithionite-quenching procedure.     

Cloning,expression, characterization,and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.